In this study we have developed a biocompatible current-conductive coating based on carbon nanotubes and bovine serum albumin and have shown its efficiency in culturing cells in vitro. We investigate the proliferation of human embryonic fibroblast (HEF) cells, which were subjected to electrical stimulation when cultured on carbon nanotube surface. A weak increase in proliferation is demonstrated at stimulating field pulses up to 100 mV. It is assumed that the transport mechanism accompanied by higher synthesis of proteins and their polymerization may increase proliferative activity at low voltages. At higher voltages the motility and spatial organization of HEF cell is observed. As a result, a novel technique of supplying the cells with electric field through a system of micro- and nanosized electrodes and a biocompatible composite have been developed.
For the past five decades electrical stimulation effect on accelerated regeneration of bone tissues, skin injury repair and targeted drug delivering has been widely investigated [
Carbon nanotubes, in particular, are one of promising nanotechnology products, and since their structure is geometrically close to collagen [
Producing a scaffold material is widely investigated in cell seeding and growing applications [
In osteoblast growth an additional possibility of delivering an electrical signal to cells through the culture medium and current-conductive composite containing nanotubes is used. In this case, a 46% increase in proliferation [
This work suggests the development of composite based on single-walled carbon nanotubes (SWNT) and bovine serum albumin (BSA) to form conductive substrates. A device for local field supply to cells through nanosized electrodes has been developed. Spatial organization and proliferation of human embryonic fibroblasts (HEF) cultured in vitro under the influence of a pulsed local electric field are studied. The research shows that by stimulating cells through nanosized electrodes at low amplitudes of electric field intensity and small signal oscillation rates proliferation raises by 26%. Whereas increasing the field strength leads to motility and HEF cell reorganization according to electric field gradient.
Most commonly the material of carbon nanotubes obtained by arch discharge or laser evaporation of graphite or by chemical decomposition of carbonaceous vapor is a powder that should be purified, filtered from undesirable impurities (such as amorphous carbon, defective nanotubes, catalyst particles), as well as functionalized, in some cases, to obtain nanotube properties required for the specific application. However in this case, preparation of solutions is needed. It is well-known that nanotubes can be dissolved in most polar and nonpolar solvents, so to dissolve SWNTs obtained in the arc discharge, it is necessary to split bunches and bundles of them which were formed in the process of growth. The most effective way to do that is additional functionalizetion of nanotubes by surfactant materials (SM), which allows a stable colloidal solution of single nanotubes of any concentration to be achieved. These SM solutions are ideal to form thin percolated films on any surfaces. By regulating nanotube concentration in the solution one can regulate the thickness of the film to be formed on the surface. As we have shown previously [
To prepare conductive composites based on nanotubes one can use their mixtures with proteins as a natural polymer providing a biocompatible substrate and improved adhesion of cells [
2.5 mg of 99.5 mass % SWNT provided by A. V. Krestinin (Institute of Problems of Chemical Physics of RAS, Moscow, Russia) were placed into a BSA aqueous solution (10 mg of BSA in 5 mL of water) and dispersed in a Branson B300 (Branson Ultrasonics, Danbury, CT, USA) ultrasonic bath (34 kHz, 50 W) for 10 hours. Typical length of nanotubes was less than 1000 nm after ultrasonic treatment. Nevertheless the CNT combines in bundles up to 5 mm long and about 10 nm in diameter. Cover-slips, 24 × 24 mm in size, 0.13 - 0.17 mm thick were preliminary mechanically washed with cotton in 2-propanol, then they were kept in 2-propanol for 15 minutes and placed in ultrasonic bath. On one of the surfaces of the cover-slip two gold contact pads, 30 nm thick, were formed by magnetron sputter deposition (Emitech K575X, Quorum Technologies, Ringmer, UK). About 25 mL of nanotube solution in albumin were applied onto this surface with a microdispenser and a thin film was made to cover the whole slip surface by rodcoating method, then the film dried for 15 minutes at 40˚C. To improve the film adhesion and conductance the structure was annealed in the air at a temperature of 150˚C for 2 minutes.
It should be noted that the samples of SWNT/BSA film obtained not only possessed conductance but also transparency within the visible range at a level of 85% - 90%.
To carry out experiments with electrical stimulation of
cells, a custom system for supplying electrical signals which comprises a 6-well plate, a signal generator and an oscilloscope has been developed. The plate cover has a breadboard with a connector fixed on it. Over each of these 6 wells two sharp needle electrodes with diameter 0.3 mm made of a 40KHNM-VI biocompatible alloy with a gold electrolytic coating (Doriva, Moscow, Russia) were placed. The electrodes were connected in pairs in 2 rows of 3 wells each to provide lead output to the signal generator connector. On the one hand, this ensured biological compatibility with the medium, on the other, the contact area of the whole surface of the needle with the cultural medium was much smaller than the area of the modified cover-slip (~40 times), which minimized the current contribution through the medium. In each set we’ve used 5 wells for experiment and one for control (
The generator can be set to arbitrarily shaped stimulatory signal, as well as to produce two independent signals in parallel. Due to the modified cultural plate design, the system was able to send a signal directly to the cells through the SWNT/BSA film. The voltage generator was connected with the plate by means of a flexible cable with the core diameter of 0.1 mm, which could be easily placed in the CO2 incubator.
For each experiment we used five cover-slips covered by SWNT/BSA film: two slips—to minimize statistical error for proliferation measurements, one—for morphology investigation, one—for microscopy analysis, one was cultured without voltage applied and used as control. Also there was one pristine cover slip with voltage applied through the medium.
HEF—human embryonic fibroblast cells were provided by the Tissue Cultures Laboratory of Ivanovskiy Institute of Virology, a Federal State Institution of the Ministry of Health and Social Development (Moscow, Russia), were cultured in the electrical stimulation unit (
After a 24 hour incubation with no voltage applied, a
signal of 5 pulses was sent with 5 ms pulse duration, 5 ms spacing between pulses, 1 s interval between pulse groups. The main pulse characteristics were chosen with previous experiments in electrical stimulation of growing fibroblasts with standard two carbon electrode unit taken into consideration [
The proliferation was defined by means of a modified MTT assay. For this purpose, the Eagle medium with 10% FBS was removed after electrical stimulation, the cover-slips were moved to a clean plate, where 1 mL of the Eagle medium and 200 ml of MTT solution (with the initial concentration of 5 mg/mL) were added. The coverslips were incubated in the thermostat with CO2 at 37˚C for 4 hours. Then the medium with MTT removed and 1 mL of DMSO was added into each well to dissolve MTT-formazan reduced by the cells. Cell precipitates with MTT-formazan on glass samples were resuspended for 5 min and the solution absorbance was measured by a Titertek Multiscan Plus photometer (Flow Laboratories, Helsinki, Finland) on a 492 nm wavelength. To achieve that, 1 mL of DMSO with the dissolved formazan was transferred to a 96-well plate, with a 100 mL per well dosage. A structure with gold electrodes and a SWNT/ BSA film applied, which was in one of the wells of a 6- well plate, but without voltage supply was used as a control.
The cover-slips with a SWNT/BSA coating were investigated with atomic force microscope Solver P47 (NT-MDT, Moscow, Russia) in a semi-contact mode before and after the cell growth. For AFM measurements samples were removed from culture medium, fixed in 2.5% glutaraldehyde for 30 minutes and washed in the phosphate buffer saline (PBS) 2 - 3 times, 2 minutes each, then dehydrated in 50%, 70% and 96% solution of ethanol for 2 min in each one. Some of the samples after electrical stimulation were stained with azur-eosine for morphology study.
First of all we have investigated the quality of film preparation methods. Looking at the surface topography shown at
uniformity of nanotube distribution in the nanomaterial array, as well as in structuring albumin itself on the nanotube surface [
Annealing at 100˚C for 2 minutes increases the conductance of the structures, but we also assume that annealing may not cause the protein denaturation, as SWNTs increase thermal stability of proteins [
Studying the morphology of HEF cells grown on glasses with SWNTs after electrical stimulation showed that the culture consisted of fibroblast-like cells with oval nuclei having big nucleoli, 1 - 4 in a nucleus, the cytoplasm was low-reticular, but there were some nanotube clusters varying in form and size (
AFM investigation was carried out on fixed cells. As nanotubes and cell membranes have different stiffness factors, it was possible to distinguish nanotubes from cells with AFM phase mode. It was found that nanotubes covered the cells on top, especially in the area of cell outgrowth formation (
The
whole, the AFM topography shows quite good adhesion of cells to the film, which indirectly confirms biological compatibility of the material.